1. Field of the Invention
The present invention relates to nanoparticles of light emissive polymers and preparation method thereof. More particularly, the present invention relates to nanoparticles of light emissive polymers, having a particle diameter, emission performance and aqueous dispersion phase which are suitable for the application as a biomolecular marker, or applications to cells or in vivo applications, so that can be used as an optical imaging contrast agent, and preparation method thereof.
2. Description of the Prior Art
In life science, fluorescence-based techniques have been widely applied for defining various basic life phenomena from molecular biology to disease diagnosis. In particular, the fluorescence-based technique uses various experimental parameters including wavelength of excitation light, wavelength of fluorescence, fluorescence lifetime or fluorescence anisotropy, as well as fluorescence intensity, thereby making it possible to multiplex signals from a plurality of targets, and provides a resolution in nanometer level and sensitivity in single molecule level.
Fluorescence-related photophysical phenomena are sensitive to the change in surrounding environments, and there are various molecular-controllable phenomena such as extinction or increase of fluorescence, generation of energy transfer, etc. resulting from interactions between homogeneous or heterogeneous materials. Such properties have been employed for the development of intelligent contrast agents for the study of interactions between biomolecules, and early diagnosis of diseases.
Fluorescent materials which have been used as an immunomarker, or a contrast agent for cells or a living body include organic fluorescent molecules [D1: Kim et al., Prog. Polym. Sci. 32:1031-1053 (2007)], fluorescent proteins [D2: Zhang et al., Nat. Rev. Mol. Cell. Bio. 3:906-918 (2002)], and inorganic quantum dots [D3: Seydack et al., Biosens. Bioelectron. 20:2454-2469 (2005), D4: Gao et al., Nat. Biotechnol. 22:969-976 (2004), and D5: Michalet et al., Science 307:538-544 (2005)].
The performances of fluorescence-based molecule detection and imaging depend on optical properties of fluorescent marking materials or contrast agents, in particular, fluorescence intensity and optical stability. The fluorescence intensity defining the sensitivity limitation of signal detection is determined by the product of the fluorescence efficiency and the absorption coefficient of a fluorescent material. In particular, the absorption coefficient for excitation light should be high in order to apply the fluorescent material to cells or a living body, where the density of excitation light is low due to scattering or absorption of the light and autofluorescence interference is severe.
The absorption coefficient for excitation light of organic fluorescent molecules or nanoparticles containing organic fluorescent molecules depends on the chemical structure of the molecule. The organic fluorescent molecules such as fluorescein, rhodamine, cyanine, etc., as described in [D6: Resch-Genger et al., Nat. Method 5:763-775 (2008)], have a molar absorption coefficient (ε) of from 2.5×104 to 2.5×105 M−1cm−1, which is not sufficient for clinical applications. In addition, organic fluorescent molecules such as coumarin, rhodamine, and the like have considerably low optical stability under continuous irradiation of excitation light, as described in Eggeling et al., Anal. Chem. 70:2651-2659 (1998) (D7).
Meanwhile, as described in D4, 5, and 6, inorganic quantum dots such as CdS, CdSe, CdTe, InP, PbS, etc. have a molar absorption coefficient (ε) of from 5×105 to 5×106 M−1cm−1, which corresponds to scores of times the value of organic fluorescent molecules, and are optically stable. However, as described in Derfus et al., Nano Lett. 4:11-18 (2004) (D8), there is a limitation in application to cells or in vivo application due to potential toxicity problems caused by a heavy metal composition.
In addition, ACS Nano 2:2415-2423 (2008) (D9) discloses π-conjugated polymers having an absorption coefficient (ε) of 1×109 M−1cm−1 or higher in a state of nanoparticle with a diameter of about 15 nm, and improved optical stability of 103 times or more compared with organic fluorescent dyes under continuous excitation light irradiation. However, π-conjugated polymers such as poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene](MEH-PPV), poly[2,5-di(3,7-dimethyloctyl)phenylene-1,4-ethynylene](PPE), poly[{9,9-dioctyl-2,7-divinylene-fluorenylene}-alt-co-{2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylene}](PFPV), poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(1,4-benzo{2,1′,3}-thiadiazole)](PFBT), etc., exhibit higher fluorescence efficiencies (Φ)) in a solution, while the fluorescence efficiencies are in general significantly declined in solid phase such as nanoparticles, thin films or the like.
Furthermore, GREENHAM et al., Nature 365:628-630 (1993) (D10) and GREENHAM et al., Chem. Phys. Lett. 241:89-96 (1995) (D11) disclosed a cyano-substituted poly(phenylene vinylene)s. These materials are π-conjugated polymers having a cyanovinylene group in their structures, which have been reported to exhibit fluorescence efficiency (Φ)) of 0.35 or higher in solid thin film. However, D10 and D11 use the π-conjugated polymers having a cyanovinylene group only for making thin films for electronic devices, and do not suggest using the same as a biomolecular marker, or optical image contrast agents to be injected into cells or a living body.